Background: There is an ongoing need to develop diverse career paths that support the vital contributions of staff scientists, research engineers, scientific officers, and other knowledge professionals in scientific discovery. Research infrastructures and core facilities have a particular need to support sustainable and diverse careers, as they either employ – if being a legal entity – or daily manage research professionals in a broad variety of roles to enable resources, services, and innovation. Methods: In 2019, a survey of the facility staff at SciLifeLab, a large national research infrastructure in Sweden, led to a recommendation for universities to develop career paths for their staff scientists. Five years later, we have conducted a survey and workshop to determine current views of infrastructure staff on career path diversity in Sweden. Results: Our results indicate there is a strong need for clarity and communication about planning and implementation of career path structures at Swedish universities, as well as opportunities to foster excellence in infrastructure staff. While the workshop participants ranked Sweden as a stable and attractive place to work and reported continuous development of their technical and service skills, the lack of recognition of this expertise presents a barrier to a sustainable career. Conclusions: We conclude that there is a need to continue advocating for increased clarity and diversity in career paths for staff scientists in Sweden, and raise the views presented by infrastructure staff on the challenges and opportunities unique to their roles.

In rod-shaped bacteria, morphological plasticity occurs in response to stress, which blocks cell division to promote filamentation. We demonstrate here that overexpression of the patatin-like phospholipase variant CapV(Q329R), but not CapV, causes pronounced sulA-independent pyridoxine-inhibited cell filamentation in the Escherichia coli K-12-derivative MG1655 associated with restriction of flagella production and swimming motility. Conserved amino acids in canonical patatin-like phospholipase A motifs, but not the nucleophilic serine, are required to mediate CapV(Q329R) phenotypes. Furthermore, CapV(Q329R) production substantially alters the lipidome and colony morphotype including rdar biofilm formation with modulation of the production of the biofilm activator CsgD, and affects additional bacterial traits such as the efficiency of phage infection and antimicrobial susceptibility. Moreover, genetically diverse commensal and pathogenic E. coli strains and Salmonella typhimurium responded with cell filamentation and modulation in colony morphotype formation to CapV(Q329R) expression. In conclusion, this work identifies the CapV variant CapV(Q329R) as a pleiotropic regulator, emphasizes a scaffold function for patatin-like phospholipases, and highlights the impact of the substitution of a single conserved amino acid for protein functionality and alteration of host physiology.

All living cells must perform essential biological processes while monitoring and responding to environmental cues. Bacteria are accessible experimental systems in which to study the function of conserved central processes. One of the most highly conserved systems between different organisms is the proteostasis network; a group of chaperones and proteases that work collectively to repair and remove damaged proteins that accumulate in living systems. In the work of this thesis, we investigate how folding machines of the proteostasis network are integrated with central biological processes in the model organism Caulobacter crescentus, and examine how these relationships change during stress.

Asymmetrically-dividing C. crescentus has previously been described to undergo aging, with the accumulation of protein aggregates in the larger stalked cell proposed to drive replicative decline in this organism. In study I, we establish C. crescentus as a model for monitoring the dynamic cellular response to protein aggregation. Using this system, we demonstrate that protein aggregates are shared during division, and do not preferentially collect in one cell type.

The ubiquitous GroESL folding machine, which provides a specialized environment for folding specific proteins, has been previously linked to the C. crescentus cell cycle through an unknown mechanism. In study II, we discover that GroESL folding is required to support division both in optimal conditions and during mild stress. Specifically, we find that GroESL supports the function of proteins that interact with the highly conserved bacterial division scaffold FtsZ, as well as proteins that direct synthesis of the peptidoglycan cell envelope layer.

In study III we investigate the functional link between GroESL folding and energy metabolism, and find that the chaperonin has a conserved role in folding respiratory and metabolic proteins, thereby supporting the central pathways these proteins function in. Furthermore, we find that GroESL protects several of these proteins from aggregation during stress.

Taken together, the work of this thesis addresses current models of prokaryotic damage segregation and aging, expands on how chaperonin folding is integrated into the essential process of division, and demonstrates a functional role for protein folding in protecting energy metabolism during stress. The findings of this research thereby provide novel insight into how fundamental biological processes interface with protein folding machines. 

Neisseria meningitidis is a gram-negative bacterium that often asymptomatically colonizes the human nasopharyngeal tract. These bacteria cross the epithelial barrier can cause life-threatening sepsis and/or meningitis. Antimicrobial peptides are one of the first lines of defense against invading bacterial pathogens. Human beta-defensin 2 (hBD2) is an antimicrobial peptide with broad antibacterial activity, although its mechanism of action is poorly understood. Here, we investigated the effect of hBD2 on N. meningitidis. We showed that hBD2 binds to and kills actively growing meningococcal cells. The lethal effect was evident after 2 h incubation with the peptide, which suggests a slow killing mechanism. Further, the membrane integrity was not changed during hBD2 treatment. Incubation with lethal doses of hBD2 decreased the presence of diplococci; the number and size of bacterial microcolonies/aggregates remained constant, indicating that planktonic bacteria may be more susceptible to the peptide. Meningococcal DNA bound hBD2 in mobility shift assays and inhibited the lethal effect of hBD2 in a dose-dependent manner both in suspension and biofilms, supporting the interaction between hBD2 and DNA. Taken together, the ability of meningococcal DNA to bind hBD2 opens the possibility that extracellular DNA due to bacterial lysis may be a means of N. meningitidis to evade immune defenses.

The highly conserved chaperonin GroESL performs a crucial role in protein folding; however, the essential cellular pathways that rely on this chaperone are underexplored. Loss of GroESL leads to severe septation defects in diverse bacteria, suggesting the folding function of GroESL may be integrated with the bacterial cell cycle at the point of cell division. Here, we describe new connections between GroESL and the bacterial cell cycle using the model organism Caulobacter crescentus. Using a proteomics approach, we identify candidate GroESL client proteins that become insoluble or are degraded specifically when GroESL folding is insufficient, revealing several essential proteins that participate in cell division and peptidoglycan biosynthesis. We demonstrate that other cell cycle events, such as DNA replication and chromosome segregation, are able to continue when GroESL folding is insuffi- cient. We further find that deficiency of two FtsZ-interacting proteins, the bacterial actin homologue FtsA and the constriction regulator FzlA, mediate the GroESL-dependent block in cell division. Our data show that sufficient GroESL is required to maintain normal dynamics of the FtsZ scaffold and divisome functionality in C. crescentus. In addition to supporting divisome function, we show that GroESL is required to maintain the flow of peptidoglycan precursors into the growing cell wall. Linking a chaperone to cell division may be a conserved way to coordinate environmental and internal cues that signal when it is safe to divide. IMPORTANCE All organisms depend on mechanisms that protect proteins from misfolding and aggregation. GroESL is a highly conserved molecular chaperone that functions to prevent protein aggregation in organisms ranging from bacteria to humans. Despite detailed biochemical understanding of GroESL function, the in vivo pathways that strictly depend on this chaperone remain poorly defined in most species. This study provides new insights into how GroESL is linked to the bacterial cell division machinery, a crucial target of current and future antimicrobial agents. We identify a functional interaction between GroESL and the cell division proteins FzlA and FtsA, which modulate Z-ring function. FtsA is a conserved bacterial actin homologue, suggesting that as in eukaryotes, some bacteria exhibit a connection between cytoskeletal actin proteins and chaperonins. Our work further defines how GroESL is integrated with cell wall synthesis and illustrates how highly conserved folding machines ensure the functioning of fundamental cellular processes during stress.

The asymmetric life cycle of Caulobacter crescentus has provided a model in which to study how protein quality control (PQC) networks interface with cell cycle and developmental processes, and how the functions of these systems change during exposure to stress. As in most bacteria, the PQC network of Caulobacter contains highly conserved ATP-dependent chaperones and proteases as well as more specialized holdases. During growth in optimal conditions, these systems support a regulated circuit of protein synthesis and degradation that drives cell differentiation and cell cycle progression. When stress conditions threaten the proteome, most components of the Caulobacter proteostasis network are upregulated and switch to survival functions that prevent, revert, and remove protein damage, while simultaneously pausing the cell cycle in order to regain protein homeostasis. The specialized physiology of Caulobacter influences how it copes with proteotoxic stress, such as in the global management of damaged proteins during recovery as well as in cell type-specific stress responses. Our mini-review highlights the discoveries that have been made in how Caulobacter utilizes its PQC network for regulating its life cycle under optimal and proteotoxic stress conditions, and discusses open research questions in this model.

Protein aggregation occurs as a consequence of perturbations in protein homeostasis that can be triggered by environmental and cellular stresses. The accumulation of protein aggregates has been associated with aging and other pathologies in eukaryotes, and in bacteria with changes in growth rate, stress resistance and virulence. Numerous past studies, mostly performed in Escherichia coli, have led to a detailed understanding of the functions of the bacterial protein quality control machinery in preventing and reversing protein aggregation. However, more recent research points toward unexpected diversity in how phylogenetically different bacteria utilize components of this machinery to cope with protein aggregation. Furthermore, how persistent protein aggregates localize and are passed on to progeny during cell division and how their presence impacts reproduction and the fitness of bacterial populations remains a controversial field of research. Finally, although protein aggregation is generally seen as a symptom of stress, recent work suggests that aggregation of specific proteins under certain conditions can regulate gene expression and cellular resource allocation. This review discusses recent advances in understanding the consequences of protein aggregation and how this process is dealt with in bacteria, with focus on highlighting the differences and similarities observed between phylogenetically different groups of bacteria.

All living cells must cope with protein aggregation, which occurs as a result of experiencing stress. In previously studied bacteria, aggregated protein collects at the cell poles and is retained throughout consecutive cell divisions only in old pole‐inheriting daughter cells, resulting in aggregation‐free progeny within a few generations. In this study we describe the in vivo kinetics of aggregate formation and elimination following heat and antibiotic stress in the asymmetrically dividing bacterium Caulobacter crescentus. Unexpectedly, in this bacterium protein aggregates form as multiple distributed foci located throughout the cell volume. Time‐lapse microscopy revealed that under moderate stress, the majority of these protein aggregates are short‐lived and rapidly dissolved by the major chaperone DnaK and the disaggregase ClpB. Severe stress or genetic perturbation of the protein quality control machinery induces the formation of long‐lived aggregates. Importantly, the majority of persistent aggregates neither collect at the cell poles nor are they partitioned to only one daughter cell type. Instead, we show that aggregates are distributed to both daughter cells in the same ratio at each division, which is driven by the continuous elongation of the growing mother cell. Therefore, our study has revealed a new pattern of protein aggregate inheritance in bacteria.